The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ∼3,500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (also known as KDM6A), JARID1C (also known as KDM5C) and SETD2 (ref. 2). These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodelling complex gene PBRM1 (ref. 4) as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology.

Colorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC.

We hypothesised that CD1d expression in renal cell carcinoma (RCC) may play a role in modifying the host immune response. Our aims were to investigate the expression of CD1d and to correlate this with histopathology and clinical outcomes in a cohort study of patients with RCC.

Opisthorchiasis is a neglected, tropical disease caused by the carcinogenic Asian liver fluke, Opisthorchis viverrini. This hepatobiliary disease is linked to malignant cancer (cholangiocarcinoma, CCA) and affects millions of people in Asia. No vaccine is available, and only one drug (praziquantel) is used against the parasite. Little is known about O. viverrini biology and the diseases that it causes. Here we characterize the draft genome (634.5 Mb) and transcriptomes of O. viverrini, elucidate how this fluke survives in the hostile environment within the bile duct and show that metabolic pathways in the parasite are highly adapted to a lipid-rich diet from bile and/or cholangiocytes. We also provide additional evidence that O. viverrini and other flukes secrete proteins that directly modulate host cell proliferation. Our molecular resources now underpin profound explorations of opisthorchiasis/CCA and the design of new interventions.

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC.

Nasopharyngeal carcinoma (NPC) has the highest metastatic potential among head and neck cancers. Distant metastasis is the major cause of treatment failure. The role of interleukin-8 (IL-8) in NPC progression remains unknown. Our multivariate survival analyses of 255 patients with NPC revealed that higher IL-8 expression in primary NPC tissue was an independent prognostic factor for overall survival, disease-free survival, and distant metastasis-free survival of the patients. In vitro study revealed that IL-8 was highly expressed in the established high-metastasis NPC clone S18 relative to the low-metastasis cells. Suppression of IL-8 by short-hairpin RNA reduced the expression of IL-8 in S18 cells and subsequently inhibited migration, invasion, and hepatic metastasis of the cells without influencing cellular growth. Overexpression of IL-8 in S26 cells resulted in increased migration, invasion, and metastasis capabilities of the cells without affecting cellular growth. Exogenous IL-8 enhanced the migration and invasion of low-metastasis CNE-2 cells in a dose-dependent manner. An epithelial-mesenchymal transition (EMT) could be induced by IL-8 in various NPC cell lines. The high level of phosphorylated AKT in S18 cells could be suppressed by knocking down IL-8 expression. Further, IL-8-promoted migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or the knock down of AKT expression by using small-interfering RNA. In summary, IL-8 serves as an independent prognostic indicator of overall survival, disease-free survival, and metastasis-free survival for patients with NPC. IL-8 promotes NPC metastasis via autocrine and paracrine means, involving activation of AKT signaling and inducing EMT in NPC cells.

Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP.

Thermal shift assay (TSA) is an increasingly popular technique used for identifying protein stabilizing conditions or interacting ligands in X-ray crystallography and drug discovery applications. Although the setting up and running of TSA reactions is a relatively simple process, the subsequent analysis of TSA data, especially in high-throughput format, requires substantial amount of effort if conducted manually. We therefore developed the Thermal Shift Assay-Curve Rapid and Automatic Fitting Tool (TSA-CRAFT), a freely available software that enable automatic analysis of TSA data of any throughput. TSA-CRAFT directly reads real-time PCR instrument data files and displays the analyzed results in a web browser. This software features streamlined data processing and Boltzmann equation fitting, which is demonstrated in this study to provide more accurate data analysis than the commonly used first-derivative method. TSA-CRAFT is freely available as a cross-operating system-compatible standalone tool ( https://sourceforge.net/projects/tsa-craft/ ) and also as a freely accessible web server ( http://tbtlab.org/tsacraft.html ).

Characterized with a high recurrence rate and low detection rate, prevention is the best approach to reduce mortality in hepatocellular carcinoma (HCC). The overexpression of Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is observed in various tumors, including HCC; and the frequent PREX2 mutations in melanoma are associated with invasiveness. We sought to identify somatic mutations and the functional changes in mutational signatures of PREX2. Genomic DNA sequencing was performed in 68 HCC samples with three types of hepatitis viral infection status: HBs Ag-positive, anti-HCV Ab-positive, and negative for any hepatitis B or C markers. Stabilities and interactions of proteins as well as cell proliferation and migration were evaluated. Fourteen non-silent point mutations in PREX2 were detected, with 16 of 68 HCC patients harboring at least one non-silent mutation. All mutant forms of PREX2, except for K400f, had an extended half-life compared with wild-type PREX2. Moreover, only the half-life of S1113R was twice that of the wild-type. PREX2 mutant-S1113R also promoted migration and activated the AKT pathway as well as impaired HectH9-mediated ubiquitination. Our study identified a gain-of-function mutation of PREX2 - S1113R in HCC. Such mutation enhanced PREX2 protein stability, promoted cell proliferation, and was associated with aggressiveness of HCC.

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

Angiosarcomas are a rare subtype of soft-tissue sarcomas which exhibit aggressive clinical phenotypes with limited treatment options and poor outcomes. In this study, we investigated the clinical relevance of the peripheral blood neutrophil-to-lymphocyte ratio (NLR) as a marker of systemic immune response, as well as its correlation with intra-tumoral immune profiles in a subgroup of cases (n = 35) using the NanoString PanCancer IO360 panel and multiplex immunohistochemistry. In the overall cohort (n = 150), angiosarcomas of the head and neck (AS-HN) comprised most cases (58.7%) and median overall survival (OS) was 1.1 year. NLR, classified as high in 78 of 112 (70%) evaluable patients, was independently correlated with worse OS (HR 1.84, 95%CI 1.18-2.87, p = 0.0073). Peripheral blood NLR was positively correlated with intra-tumoral NLR (tNLR) (Spearman's rho 0.450, p = 0.0067). Visualization of tumor-infiltrating immune cells confirmed that tNLR scores correlated directly with both neutrophil (CD15+ cells, rho 0.398, p = 0.0198) and macrophage (CD68+ cells, rho 0.515, p = 0.0018) cell counts. Interestingly, tNLR correlated positively with oncogenic pathway scores including angiogenesis, matrix remodeling and metastasis, and cytokine and chemokine signaling, as well as myeloid compartment scores (all p < 0.001). In patients with documented response assessment to first-line chemotherapy, these pathway scores were all significantly higher in non-responders (47%) compared to responders. In conclusion, systemic and local immune responses may inform chemotherapy response and clinical outcomes in angiosarcomas.

Peritoneal carcinomatosis (PC) present a ubiquitous clinical conundrum in all intra-abdominal malignancies. Via functional and transcriptomic experiments of ascites-treated PC cells, we identify STAT3 as a key signaling pathway. Integrative analysis of publicly available databases and correlation with clinical cohorts (n = 7,359) reveal putative clinically significant activating ligands of STAT3 signaling. We further validate a 3-biomarker prognostic panel in ascites independent of clinical covariates in a prospective study (n = 149). Via single-cell sequencing experiments, we uncover that PAI-1, a key component of the prognostic biomarker panel, is largely secreted by fibroblasts and mesothelial cells. Molecular stratification of ascites using PAI-1 levels and STAT3 activation in ascites-treated cells highlight a therapeutic opportunity based on a phenomenon of paracrine addiction. These results are recapitulated in patient-derived ascites-dependent xenografts. Here, we demonstrate therapeutic proof of concept of direct ligand inhibition of a prognostic target within an enclosed biological space.

Treatment efficacy of androgen deprivation therapy with radical prostatectomy for intermediate- to high-risk prostate cancer is less well-studied. The NEAR trial is a single-arm, phase II investigation of neoadjuvant apalutamide monotherapy and radical prostatectomy (RP) in the treatment of D'Amico intermediate- and high-risk prostate cancer (NCT03124433).

Angiosarcomas are rare malignant tumors of the endothelium, arising commonly from the head and neck region (AS-HN) and recently associated with ultraviolet (UV) exposure and human herpesvirus-7 infection. We examined 81 cases of angiosarcomas, including 47 cases of AS-HN, integrating information from whole genome sequencing, gene expression profiling and spatial transcriptomics (10X Visium). In the AS-HN cohort, we observed recurrent somatic mutations in CSMD3 (18%), LRP1B (18%), MUC16 (18%), POT1 (16%) and TP53 (16%). UV-positive AS-HN harbored significantly higher tumor mutation burden than UV-negative cases (p = 0.0294). NanoString profiling identified three clusters with distinct tumor inflammation signature scores (p < 0.001). Spatial transcriptomics revealed topological profiles of the tumor microenvironment, identifying dominant but tumor-excluded inflammatory signals in immune-hot cases and immune foci even in otherwise immune-cold cases. In conclusion, spatial transcriptomics reveal the tumor immune landscape of angiosarcoma, and in combination with multi-omic information, may improve implementation of treatment strategies.

Myoblast transfer therapy (MTT) is a technique to replace muscle satellite cells with genetically repaired or healthy myoblasts, to treat muscular dystrophies. However, clinical trials with human myoblasts were ineffective, showing almost no benefit with MTT. One important obstacle is the rapid senescence of human myoblasts. The main purpose of our study was to compare the various methods for scalable generation of proliferative human myoblasts.

Previous genome-wide association studies (GWAS) have identified six risk loci for renal cell carcinoma (RCC). We conducted a meta-analysis of two new scans of 5,198 cases and 7,331 controls together with four existing scans, totalling 10,784 cases and 20,406 controls of European ancestry. Twenty-four loci were tested in an additional 3,182 cases and 6,301 controls. We confirm the six known RCC risk loci and identify seven new loci at 1p32.3 (rs4381241, P=3.1 × 10-10), 3p22.1 (rs67311347, P=2.5 × 10-8), 3q26.2 (rs10936602, P=8.8 × 10-9), 8p21.3 (rs2241261, P=5.8 × 10-9), 10q24.33-q25.1 (rs11813268, P=3.9 × 10-8), 11q22.3 (rs74911261, P=2.1 × 10-10) and 14q24.2 (rs4903064, P=2.2 × 10-24). Expression quantitative trait analyses suggest plausible candidate genes at these regions that may contribute to RCC susceptibility.

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.

Von Hippel-Lindau (VHL) disease is a rare autosomal dominant syndrome that is the main cause of inherited clear-cell renal cell carcinoma (ccRCC), which generally occurs in the form of multiple recurrent synchronized tumors. Affected patients are carriers of a germline mutation in the VHL tumor suppressor gene. Somatic mutations of this gene are also found in sporadic ccRCC and numerous pan-genomic studies have reported a dysregulation of microRNA (miRNA) expression in these sporadic tumors. In order to investigate the molecular mechanisms underlying the pathogenesis of VHL-associated ccRCC, particularly in the context of multiple tumors, the present study characterized the mRNA and miRNA transcriptome through an integrative analysis compared with sporadic renal tumors. In the present study, two series of ccRCC samples were used. The first set consisted of several samples from different tumors occurring in the same patient, for two independent patients affected with VHL disease. The second set consisted of 12 VHL-associated tumors and 22 sporadic ccRCC tumors compared with a pool of normal renal tissue. For each sample series, an expression analysis of miRNAs and mRNAs was conducted using microarrays. The results indicated that multiple tumors within the kidney of a patient with VHL disease featured a similar pattern of miRNA and gene expression. In addition, the expression levels of miRNA were able to distinguish VHL-associated tumors from sporadic ccRCC, and it was identified that 103 miRNAs and 2,474 genes were differentially expressed in the ccRCC series compared with in normal renal tissue. The majority of dysregulated genes were implicated in 'immunity' and 'metabolism' pathways. Taken together, these results allow a better understanding of the occurrence of ccRCC in patients with VHL disease, by providing insights into dysregulated miRNA and mRNA. In the set of patients with VHL disease, there were few differences in miRNA and mRNA expression, thus indicating a similar molecular evolution of these synchronous tumors and suggesting that the same molecular mechanisms underlie the pathogenesis of these hereditary tumors.

Next generation sequencing technology has revolutionized the study of cancers. Through matched normal-tumor pairs, it is now possible to identify genome-wide germline and somatic mutations. The generation and analysis of the data requires rigorous quality checks and filtering, and the current analytical pipeline is constantly undergoing improvements. We noted however that in analyzing matched pairs, there is an implicit assumption that the sequenced data are matched, without any quality check such as those implemented in association studies. There are serious implications in this assumption as identification of germline and rare somatic variants depend on the normal sample being the matched pair. Using a genetics concept on measuring relatedness between individuals, we demonstrate that the matchedness of tumor pairs can be quantified and should be included as part of a quality protocol in analysis of sequenced data. Despite the mutation changes in cancer samples, matched tumor-normal pairs are still relatively similar in sequence compared to non-matched pairs. We demonstrate that the approach can be used to assess the mutation landscape between individuals.

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase EGLN2 [corrected] a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of EGLN2 [corrected] in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.